Development of an ENPP1 Fluorescence Probe for Inhibitor Screening, Cellular Imaging, and Prognostic Assessment of Malignant Breast Cancer

Ectonucleotide
pyrophosphatase/phosphodiesterase 1 (ENPP1) is a type II transmembrane
glycoprotein that is involved in bone metabolism and insulin resistance,
hydrolyzes 2′,3′-cGAMP (a STING ligand that promotes
innate immunity), and is associated with cancer stemness in breast
cancers and glioblastoma. Therefore, ENPP1 is considered a candidate
therapeutic target and/or biomarker for early diagnosis of malignant
tumors. In this study, we designed and synthesized a sensitive ENPP1
fluorescence probe, Tokyo Green (TG) mAMP. We used it to screen a
chemical library for non-phosphate ENPP1 inhibitors. Structural optimization
of a selected hit afforded a potent and specific ENPP1 inhibitor.
We further found that ENPP1 mRNA expression in tissue
samples from patients with triple-negative breast cancer was significantly
inversely related to recurrence-free survival (RFS) and overall survival
(OS), and TG-mAMP assay revealed a significant difference in ENPP1
activity between ENPP1 high-expressing and ENPP1 low-expressing samples. Our results suggest that TG-mAMP
assay might be a rapid and inexpensive tool for predicting the prognosis
of patients with malignant breast cancers.